Development of a novel zebrafish xenograft model in ache mutants using liver cancer cell lines

Acetylcholinesterase (AChE), an enzyme responsible for degradation of acetylcholine, has been identified as a prognostic marker in liver cancer. Although in vivo Ache tumorigenicity assays in mouse are present, no established liver cancer xenograft model in zebrafish using an ache mutant background exists. Herein, we developed an embryonic zebrafish xenograft model using epithelial (Hep3B) and mesenchymal (SKHep1) liver cancer cell lines in wild-type and achesb55 sibling mutant larvae after characterization of cholinesterase expression and activity in cell lines and zebrafish larvae. The comparison of fluorescent signal reflecting tumor size at 3-days post-injection (dpi) revealed an enhanced tumorigenic potential and a reduced migration capacity in cancer cells injected into homozygous achesb55 mutants when compared with the wild-type. Increased tumor load was confirmed using an ALU based tumor DNA quantification method modified for use in genotyped xenotransplanted zebrafish embryos. Confocal microscopy using the Huh7 cells stably expressing GFP helped identify the distribution of tumor cells in larvae. Our results imply that acetylcholine accumulation in the microenvironment directly or indirectly supports tumor growth in liver cancer. Use of this model system for drug screening studies holds potential in discovering new cholinergic targets for treatment of liver cancers.

. Ache western blot in liver cancer cell lines and zebrafish brain, liver tissues. Liver cancer cell lines (and a colon carcinoma cell line SW620) were grown to confluency in 10 cm culture dishes in respective culture media and collected by scraping. Total protein was isolated using NP40 lysis buffer. Zebrafish liver and brain tissues dissected under stereo microscope and homogenates were prepared by passing through syringe 20 times in NP40 lysis buffer, followed by centrifugation and supernatant collection. Total protein amounts measured by Bradford assay and 50 µg protein was loaded in 5x loading buffer containing a final amount of 1% β-mercaptoethanol. Proteins were separated using 10% SDS-PAGE. Proteins were transferred to PVDF membrane by wettransfer. Membrane was blocked overnight in 5% skim milk-TBST buffer at 4 o C. Goat polyclonal Ache (Abcam-ab31276) antibody was diluted in above described blocking buffer at a final 1:400 concentration and membrane was incubated overnight at 4 o C in this solution. After TBST washes, membrane was further incubated in secondary HRP conjugated goat polyclonal to rabbit IgG (H+L) at a final 1:5000 dilution in blocking buffer for 1 hr at room temperature. Following TBST washes, signal was developed using ECL+ (GE Healthcare) system. For equal loading, a rabbit polyclonal β-tubulin antibody was used. Figure comes from two separately run gels on the left and right handsights separated by a vertical solid white line, accordingly the same membranes were incubated with the above mentioned two different antibodies consequently following harsh membrane stripping.
Ache protein was detected in all studied HCC cell lines. There is low expression in HepG2, Huh7 and Snu387 cell lines. Ache antibody did not recognize zebrafish ache proteins due to lack of a respective band for zebrafish tissue homogenate sample wells.

Supplementary Figure 2. ache expression analysis in AB line at different developmental stages. (a)
ache expression was analyzed by using ache coding sequence spanning RT-PCR primers at 1, 2, 3, 6, 12, 24, and 48hpf. Amplification was performed for 35 cycles. cDNA was prepared from 1 microgram of total RNA. ache expression started between 12-24hpf. At 12 hpf there was a non-specific PCR band different than the expected amplicon size of 1905bp. (b) qPCR quantitative ache expression analysis with a new set of cDNA (N = 1 experiment; n = 20 embryos/group). In addition to the above stages RNA from ache-/-and +/? larvae at 72 hpf were included into analysis (N = 1 experiment; n = 20 embryos/group). At 72hpf ache expression was further increased compared to 48hpf expression level. elfa was used as housekeeping gene. ache in situ hybridization probes used to detect mRNA localization at (c, c') 24 hpf where myotome (arrowheads) was stained; at (d, d') 48 hpf where ventrorostral (arrow) and ventrocaudal clusters (arrowhead) in the inner brain were labeled. my: myotome, vrc: ventrorostral cluster, vcc: ventrocaudal cluster. Figure 3. ache sb55 mutant selection and characterization. Embryos obtained from ache sb55 heterozygote parental fish were grouped using "tail-test". (a) At 3 dpf, ache wild type siblings were able to swim following consecutive disturbance (a'). (b) Whereas, ache sb55 homozygous mutant fish were paralyzed due to excess ACh accumulation and could not move after touching their tails (b').

Supplementary Figure 4. Comparison of the human DNA amount (pg) across the AluYb8 Ct values.
Serial dilutions from 10 ng to 0.01 pg of human DNA mixed with 10 ng of wild type zebrafish DNA were used for detection of AluYb8 using qPCR.

Suplementary Figure 5. The linear regression analysis of the zebrafish DNA amount measured with ache primers from embryos injected with Hep3B or SKHep1
. Ct values of the main primer (added to 1) was used for the homozygous ache+/+ or ache-/-larvae while mean of both primers was used for the heterozygous larvae (r = -0.43 and P = 9.46e-04 n=55 for SKHep1; r = -0.57 and P = 5.37e-07 n= 66 for Hep3B).